Bruton's Tyrosine Kinase (BTK) is member of the Tec family of non-receptor tyrosine kinases that is critically important for the growth, differentiation and activation of B-cells, myeloid cells, and mast cells. The BTK gene is located at cytogenetic band Xq21.33-q22 and comprises 19 exons, spanning 37 kb, encoding the full length BTK protein. The central role of BTK in B cell function is underscored by the human disease X-linked agammaglobulinemia, or Bruton's agammaglobulinemia, which is caused by loss of function mutations in BTK. These mutations result in the virtual absence of all B cells and immunoglobulins, leading to recurrent bacterial infections.
BTK is essential to B-cell receptor (BCR) signaling and in knockout mouse models, its mutation has a B cell-specific phenotype. BTK protein and mRNA are significantly over-expressed in chronic lymphocytic leukemia (CLL) compared with normal B-cells. Although BTK is not always constitutively active in CLL cells, B-cell receptor (BCR) or CD40 signaling is accompanied by effective activation of this pathway. BTK activity is involved in the disease progression of B-cell malignancies, such as Non-Hodgkin's Lymphomas, such as chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM).
BTK is activated by membrane localization stimulated by PIP3 (phosphatidlinositol-3,4,5-triphosphate) generation and bonding to the BTK pleckstrin homology (PH) domain, and transphosphorylation of Tyr-551 by Src family kinases. Activated BTK is involved in the phosphorylation of a number of signaling molecules involved in the PLCγ (phospholipase c gamma), JNK (c-Jun NH2-terminal kinase) and p38 MAPK pathways, leading to Ca2+ mobilization, mRNA stabilization and the induction of NF-κB and AP-1 transcription factors. BTK activity is negatively regulated by a number of proteins including inhibitor of BTK (IBTK), Sab and c-Cbl. During antigenic challenge, the classical NF-κB pathway is strongly activated by B-cell receptor signaling, via formation of a “CBM” signaling complex consisting of CARD11, MALT1, and BCL10. The CBM lies downstream of PLCγ activation of BTK. The CBM pathway is pathologically altered in several lymphoma subtypes; mutations in CARD11 have been found to constitutively activate downstream NF-κB signaling.
Chronic lymphocytic leukemia (CLL) remains the most common leukemia of adults, and is incurable. Although generally considered indolent, most patients will ultimately die of the disease. Current therapies are effective in inducing initial remission in most patients who can tolerate them, but these therapies are not curative, and resistance ultimately develops.
Ibrutinib (PCI-32765 (Pharmacyclics, Sunnyvale, Calif.)) is a potent covalent kinase inhibitor that targets BTK, binding covalently to Cys-481 in the active site of BTK, resulting in inhibition of kinase activity with IC50 0.5 nM. (See J. R. Brown, PCI-32765, the First BTK (Bruton's Tyrosine Kinase) Inhibitor in Clinical Trials, Curr Hematol Malig Rep. 2013 March; 8(1): 1-6, incorporated herein by reference.) Ibrutinib, which has been approved by the USFDA as a treatment for mantle cell lymphoma and chronic lymphocytic leukemia, causes rapid nodal reduction and response associated with rapid increase in lymphocytosis, which then returns to baseline over time. Ibrutinib has also been demonstrated to be efficacious in certain autoimmune diseases such as arthritis and lupus. Patients with chronic lymphocytic leukemia (CLL) that develop resistance to BTK inhibitors are typically positive for histologic transformation or mutations in BTK or phospholipase c gamma 2 (PLCγ2). Mutations in BTK at the C481S hotspot alter the active site of the mutant BTK to the effect that ibrutinib is reversibly bound. PLCγ2 is downstream of BTK in the B-cell signaling pathway; mutations in PLCγ2 at either of the R665W, L845F, or S707Y hotspots result in a constitutively activated PLCγ2. (See, e.g., U.S. Patent Publ. 2015/0184249 A1, which is incorporated herein by reference.)
Bruton tyrosine kinase (BTK) inhibitors like ibrutinib have demonstrated high clinical response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL) including refractory patients to conventional therapy or patients with tumor protein p53 (TP53) mutations. Patients who develop resistance to ibrutinib therapy typically have mutations in either BTK or phospholipase c γ 2 (PLCγ2). Mutations in BTK at the C481S hotspot alter the BTK binding site rendering it reversible to binding ibrutinib resulting in ineffective therapeutic results. Alternatively, mutations in PLCγ2, which is immediately downstream of BTK in the B-Cell receptor signaling pathway, result in a gain of function and BTK independent B-Cell Receptor activation. While the emergence of these mutations has been reported to be associated with resistance to therapy, little is known about the development of these resistance mutations throughout the course of therapy. In clinical trials of CLL patients on BTK inhibitor (BTKi) therapy, whole exome sequencing with next-generation sequencing (NGS) has typically been used to detect specific mutations in BTK or PLCg2 genes. Therefore, accurate, high-sensitivity assays that can be run in large volumes in a clinical setting are a necessity to further understand the relationship between the appearance of a mutation and the development of resistance to therapy and clinical progression.
Since the introduction of next-generation sequencing (NGS) technology, there has been a major transformation in the way researchers extract genetic information from biological systems, opening the way to expanded insight about the genome, transcriptome, and epigenome of any species. This ability has catalyzed a number of important breakthroughs, advancing fields from human disease research to agriculture and evolutionary science.
In principle, the concept behind NGS technology is similar to capillary electrophoresis (CE)-based Sanger sequencing: the bases of a small fragment of DNA are sequentially identified from signals emitted as each fragment is re-synthesized from a DNA template strand. NGS extends this process across millions of reactions in a massively parallel fashion, without being limited to a single or a few DNA fragments. This advance enables rapid sequencing of large strings of DNA base pairs spanning entire genomes, with the latest instruments being capable of producing hundreds of gigabases of data in a single sequencing run.
With the advent of NGS, sequencing and testing for mutations has become a standard procedure in the diagnosis and management of patients with cancer. Screening for various mutations in cancer tissue provides a means for predicting prognosis and for determining therapy. Precision medicine and targeted therapy depends on the detection of molecular abnormalities and selecting therapy that target these molecular abnormalities.
While NGS has provided a great tool for detecting mutations with a sensitivity in the range of 5%, it remains less sensitive for the detection of mutations that present in less than 5% of the analyzed DNA. This is particularly the case when attempting to analyze peripheral blood plasma. Accordingly, the need remains for method for improving the sensitivity of NGS for purposes of detecting low-occurrence mutations.
Wild-type blocking polymerase chain reaction (WTB-PCR) followed by Sanger sequencing has demonstrated high sensitivity and versatility in the detection of low percentage mutant DNA. By adding a short (10-12 mer) inaccessible [locked or bridged nucleic acid (LNA or BNA)] oligonucleotide, complementary to wild-type hotspot loci, amplification of the wild-type (WT) allele is inhibited, leading to experimentally driven positive selection for mutant alleles. Because a single nucleotide mismatch in the LNA/BNA-DNA hybrid greatly decreases its melting temperature, only mutant template DNA is free to complete its extension. Therefore, WT DNA is amplified linearly but mutant DNA is amplified exponentially. BNA is a third generation nucleic acid analog with excellent mismatch discriminating power and is considered more potent in blocking. Its strong nuclease resistant properties coupled with a 3′ phosphate also prevents amplification of the wild-type DNA and selectively amplifies mutant DNA. The resulting WTB-PCR product can then be sequenced by traditional Sanger sequencing methods. We also theorized that the same principle could be applied to NGS library preparation.
While WTB-PCR/Sanger sequencing or WTB-PCR/NGS can provide accurate, high-sensitivity mutation analysis, spatial sampling bias in patients with lymphomas or CLL with few circulating tumor cells and lymph node or organ involvement could potentially lead to false negatives. This is particularly relevant when tumor heterogeneity is considered. The presence of a mutation in a subclone of the tumor cells can be easily missed if the subclone is not circulating or patchy in bone marrow—if bone marrow aspiration is used. In patients with hematologic diseases, the peripheral blood (PB) plasma has been demonstrated to be enriched for tumor-specific DNA, RNA, and proteins. This is especially true for the DNA of the more aggressive subclone. Testing cell-free DNA (cfDNA) from plasma or serum may therefore provide greater sensitivity for detecting resistance mutations than cellular DNA from PB.
In order to better understand the development of these resistance mechanisms in patients with CLL, high sensitivity testing is needed. The present invention is directed to a method for such testing.